Infectious diseases represent a major burden on healthcare resources. In addition, the emergence of antibiotic resistant bacterial strains and concerns of bioterrorism have increased the need to understand the normal defense mechanisms that combat pathogens. Phagocytosis, the process of cellular engulfment of microbes into a newly formed membrane-limited organelle, the phagosome, is an essential component of the innate immune defense system. It directs bacteria into a highly hydrolytic and acidic environment, the mature phagolysosome, which limits replication and destroys the engulfed organism. An additional component of host defense is inflammatory signaling, which is triggered by pattern recognition receptors (PRRs), including the transmembrane Toll-like receptors (TLRs) and the cytosolic NOD-like receptors (NLRs). We have observed that certain pathogens, such as S. aureus, exhibit absolute dependence upon the phagosome to initiate immune activation: signaling cannot be triggered from the cell surface and bacteria must be delivered into a mature phagolysosome for induction of proinflammatory cytokines response. We hypothesize that for these pathogens the phagosome plays an active and essential role in sensing and coordinating innate immune signaling by both TLRs and NLRs. Our proposal will investigate the cellular mechanism of phagosome-dependent innate immune activation by determining the role of this organelle in generation of TLR and NLR ligands, recruitment of NLRs to the phagosome and transport of NLR ligands to the cytosol. We anticipate that the completion of these aims will further our understanding of the innate immune mechanisms that recognize, and respond to, microorganisms. These results will be relevant to understanding the host defense to a number of pathogens. PUBLIC HEALTH RELEVANCE Infectious diseases represent a major burden on healthcare resources. In addition, the emergence of antibiotic resistant bacterial strains and concerns of bioterrorism have increased the need to understand the normal defense mechanisms that combat pathogens. We have observed that response to certain bacteria, such as the remerging pathogen S. aureus, occurs only after cells called `phagocytes' eat the bacteria. Moreover, this process of phagocytosis is essential for our host defense to be fully activated. We propose to study the role phagocytosis in sensing of bacterial invasion and induction of our normal immune defense.